Toxic Elements in Beans from Zhejiang, Southeast China: Distribution and Probabilistic Health Risk Assessment.

This study described the distribution of As, Cd, Cr, Hg, and Pb in 692 bean samples from Zhejiang province, southeast China, and estimated the health risk using Monte Carlo simulation. The average levels of As, Cd, Cr, Hg, and Pb were 0.0349, 0.0379, 0.246, 0.0019, and 0.0246 mg kg-1. Correlation analyses showed very strong positive correlations for Cd-Pb in kidney beans and mung beans, Cd-As in black beans, and Pb-As in red beans. The target hazard quotients (THQs) were adopted for non-carcinogenic risk assessment, and THQs at the 50th percentile were all less than 1, indicating that there are no deleterious effects from rice exposure to these elements. When evaluating THQ for multiple elements, the certainty with a hazard index (HI) greater than 1 for children was 12.64%, for teens 11.54%, and for adults 1.01%. The sensitivity analysis reveals that the concentration of Cd in beans and ED (exposure duration) are the main principal factors that contributed to the total risk. The mean carcinogenic risks for children, teens, and adults were all less than 1 × 10-4, indicating no potential carcinogenic risk. Despite that, the routine monitoring of these elements, especially for Cd should be continued.

Heavy metals accumulation in bivalve mollusks collected from coastal areas of southeast China.

The distribution of six heavy metal and metalloids (As, Cd, Cr, Hg, Ni and Pb) was analyzed in 597 bivalve mollusks (8 species) collected from coastal areas of southeast China. Target hazard quotient, total hazard index, and target cancer risk were calculated to evaluate potential human health risks from bivalve consumption. The mean concentrations of As, Cd, Cr, Hg, Ni and Pb were 1.83, 0.581, 0.111, 0.0117, 0.268 and 0.137 mg kg-1 wet weight in bivalves. The average estimated daily intakes for As, Cd, Cr, Hg, Ni and Pb were 1.156, 0.367, 0.07, 0.007, 0.167 and 0.087 µg kg-1 body weight/day. Health risk assessment showed that there was no non-carcinogenic health risk to general residents to these metals from consumption of bivalves. Cd intake through mollusks posed a potential cancer risk. Accordingly, regular monitoring for heavy metals, especially Cd is recommended with respect to potential contaminant on marine ecosystems.

Distribution of Cadmium in Fresh Vegetables Marketed in Southeast China and Its Dietary Exposure Assessment.

This study investigated concentrations of cadmium (Cd) in 2465 vegetable samples (52 species) from 2018 to 2022 and estimated the associated health risk for local consumers. The average concentration of Cd was 0.035 mg kg-1, and the percentage of samples exceeding the Chinese maximum allowed concentration was 3.89% (96/2465). The top five species with highest Cd levels were Lilium brownii F (0.182 mg kg-1), Allium chinense G (0.117 mg kg-1), Allium macrostemon Bunge (0.105 mg kg-1), Colocasia esculenta (0.064 mg kg-1), and Amaranthus tricolor L (0.054 mg kg-1). Bulb vegetables had a higher relative accumulation of Cd compared to other vegetables. The levels of Cd in vegetables varied significantly across sampling areas and years. The mean estimated daily intake (EDI) of cadmium through consumption of vegetables was 0.519 µg kg-1 bw per day for adults and 0.217 µg kg-1 bw per day for children. The target hazard quotients (THQs) were all less than the threshold of 1 for both adults and children. This indicates that there is low health risk for Cd through vegetable consumption. However, routine monitoring of Cd levels in food is still crucial to ensure food safety and protect public health.

Screening of multi-class antibiotics in pork meat by LC-Orbitrap-MS with modified QuEChERS extraction.

The quantification capability of high resolution mass spectrometry is of great interest to analysts. We described a method for analysis of multi-class antibiotics in pork meat by UPLC-quadrupole (Q)-Orbitrap-MS. The QuEChERS approach with a clean-up step using a sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation, and 37 antibiotics including beta-lactams, tetracyclines, sulfonamides, fluoroquinolones and macrolides were analyzed. The Q-Orbitrap method showed high sensitivity with limits of detection (LODs) ranging from 0.8 µg kg-1 to 2.9 µg kg-1. The method was further validated by intra and inter-day tests with fortified samples. Recovery (85-105.6%) and precision values (RSDs < 15%) for all analytes were obtained. The result indicates that UPLC-Q-Orbitrap-MS coupled with QuEChERS preparation can serve as a routine method for multi-class antibiotic analysis in pork meat.

Abnormal resting-state functional connectivity in posterior cingulate cortex of Parkinson's disease with mild cognitive impairment and dementia.

OBJECTIVE: To investigate changes in the functional connectivity (FC) pattern in the posterior cingulate cortex (PCC) of Parkinson's disease (PD) patients with mild cognitive impairment and dementia by employing resting-state functional magnetic resonance imaging (RS-fMRI). METHODS: Twenty-seven PD patients with different cognitive status and 9 healthy control subjects (control group) were enrolled for RS-fMRI. The RS-fMRI data were analyzed with DPARSF and REST software. Regions with changed functional connectivity were determined by the seed-based voxelwise method and compared between groups. Correlation between the intensity of FC and the MoCA scores of PD group was analyzed. RESULTS: Parametric maps showed statistical increases in PCC functional connectivity in PD-MCI patients and decreases in PCC connectivity in PDD patients. The latter group of patients also showed evidence for increased connectivity between prefrontal cortices and posterior cerebellum. A significant positive correlation was found between the MoCA scores and the strength of PCC connectivity in the angular gyrus and posterior cerebellum and a negative correlation between MoCA scores and PCC connectivity in all other brain regions. CONCLUSION: When patients transition from PD-NCI to PD-MCI, there appears to be an increase in functional connectivity in the PCC, suggesting an expansion of the cortical network. Another new network (a compensatory prefrontal cortical-cerebellar loop) later develops during the transition from PD-MCI to PDD.

Dual inhibition of BRD4 and PI3K-AKT by SF2523 suppresses human renal cell carcinoma cell growth.

Bromodomain-containing protein 4 (BRD4) and PI3K-AKT are both important for renal cell carcinoma (RCC) development and progression. SF2523 is a BRD4 and PI3K-AKT dual inhibitor. The present study demonstrated that SF2523 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. SF2523 induced activation of caspase and apoptosis in RCC cells. Further, SF2523 disrupted RCC cell cycle progression and inhibited cell migration in vitro. At the signaling level, SF2523 in-activated PI3K-AKT-mTOR, and downregulated BRD4-dependent proteins, Bcl-2 and Myc, in RCC cells. Remarkably, SF2523 was more efficient than Wortmannin (the PI3K inhibitor) and JQ1 (the BRD4 specific inhibitor) in killing RCC cells. In vivo, SF2523 administration at well-tolerated doses suppressed 786-O xenograft tumor growth in severe combined immunodeficient (SCID) mice. Together, our results suggest that concurrent blockage of BRD4 and PI3K-AKT signalings by SF2523 efficiently inhibits RCC cell growth in vitro and in vivo.

Quantification of 16 ß-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-Orbitrap-MS with parallel reaction monitoring.

A method is described for the analysis of 16 ß-lactams in chicken muscle by UPLC-quadrupole(Q)-Orbitrap-MS with parallel reaction monitoring (PRM). QuEChERS approach includes clean-up step by sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation. Q-Orbitrap with PRM showed high sensitivity with limits of detection (LODs) ranged from 0.01µgkg-1 to 0.35µgkg-1. The method was further validated by intra- and inter-day test with spiking levels less than MRLs (maximum residue limits, the European Union). Recovery (83-112%) and precision values (RSDs <15%) for all studied analytes were obtained. The result indicates that UPLC-Q-Orbitrap coupled with QuEChERS preparation can serve as a routine quantification method for ß-lactam residues in chicken muscles.

Analysis of LDLR variants from homozygous FH patients carrying multiple mutations in the LDLR gene.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is an autosomal dominant disease with widespread global prevalence that partially accounts for the high prevalence of premature coronary heart disease. Although the majority of research on FH has focused on single heterozygous LDLR mutations, there have been limited reports of double LDLR mutations on the same chromosome. The aim of this study was to gain insight into the clinical consequences of the presence of multiple mutations in the LDLR gene. METHODS: DNA from two clinical homozygous FH patients and their relatives was analysed using targeted exome sequencing and DNA resequencing. Functional characterization of novel variants was performed by Western blot, flow cytometry and confocal microscopy. RESULTS: Proband 1 carried p.Q12X, NTDA (p.N276T and c.892delA) mutations in LDLR, and Proband 2 carried c.971delG, GSDN (p.G77S + D601N). Results showed that p.Q12X, c.892delA, and c.971delG are non-functional LDLR variants. Conversely, N276T and G77S are non-pathogenic variants. Interestingly, while D601N alone only slightly diminishes LDLR activity, its co-presence with the non pathogenic p.G77S mutation results in a more strongly pathogenic variant with LDLR activity reduced by 40%. One of the double mutants, NTDA, is as non functional as c.892delA alone. The other double mutant, GSDN, is more severe than either of the component single mutants. CONCLUSIONS: An early gene screening and laboratory functional verification of LDLR activity is of vital importance to enable a definite FH diagnosis. Functional verification is also necessary for prenatal and postnatal care in patients with FH.

microRNA-302c-3p inhibits renal cell carcinoma cell proliferation by targeting Grb2-associated binding 2 (Gab2).

The expression and biological function of Grb2-associated binding 2 (Gab2) in renal cell carcinoma (RCC) cells was tested here. We showed that Gab2 expression was significantly elevated in human RCC tissues and RCC cells. It was correlated with over-activation of Akt and downregulation of microRNA-302c-3p ("miR-302c-3p"), a putative Gab2-targeting microRNA. Knockdown of Gab2 inhibited Akt activation and 786-O RCC cell proliferation. Reversely, forced over-expression of Gab2 led to Akt hyper-activation to facilitate 786-O cell proliferation. Exogenous expression of miR-302c caused Gab2 downregulation, Akt inhibition and 786-O cell proliferation inhibition. On the other hand, miR-302c-3p depletion by expressing its anti-sense ("antagomiR-302c") led to Gab2 upregulation, Akt activation and increased 786-O cell proliferation. Significantly, miR-302c-3p failed to affect the proliferation of 786-O cells with shRNA-depleted Gab2. Together, we suggest that miR-302c-3p depletion in human RCC cells leads to Gab2 over-expression, Akt hyper-activation and cell proliferation.

Concurrent inhibition of mTORC1 and mTORC2 by WYE-687 inhibits renal cell carcinoma cell growth in vitro and in vivo.

Mammalian target of rapamycin (mTOR)in renal cell carcinoma (RCC) represents a valuable oncotarget for treatment. We here tested the potential anti-RCC activity by a novel mTOR kinase inhibitor WYE-687in vitro and in vivo.WYE-687 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. Yet, it was non-cytotoxic toHK-2 tubular epithelial cells.WYE-687 provoked caspase-dependent apoptosis in the RCC cells. At the molecular level, WYE-687 almost completely blocked mTORC1 (p-S6K1 and p-S6) and mTORC2 (p-Akt Ser 473) activation in both 786-Ocells and primary human RCC cells, where it downregulated both hypoxia-inducible factor (HIF)-1α and HIF-2α expression. Significantly, oral administration of WYE-687 potently suppressed786-O tumor xenograft growth in nude mice. mTORC1/2 activation and HIF-1α/2α expression were also remarkably downregulated in WYE-687-treated tumor tissues. Thus, our preclinical results imply that WYE-687 may have important translational value for the treatment of RCC.

Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation.

Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation.

Characterization of the unique Chinese W483X mutation in the low-density lipoprotein-receptor gene in young patients with homozygous familial hypercholesterolemia.

BACKGROUND: Recent guidelines suggest that more attention should be focused on children with homozygous familial hypercholesterolemia (HoFH). China may have 3.8 million potential FH patients, but there are limited data focused on HoFH children. OBJECTIVE: We systematically analyzed the characteristic phenotype and the relationship between the genotype and the phenotype in HoFH children with the unique Chinese W483X mutation in the low-density lipoprotein (LDL)-receptor gene. METHODS: A systematic retrospective analysis of the lipid and cardiovascular characteristics of HoFH patients in the atherosclerosis clinic of Beijing Anzhen Hospital was performed. The W483X mutation was confirmed using DNA sequencing of the patients and their parents. RESULTS: Two HoFH and 9 compound heterozygous patients (mean age = 14.7 years) with 2 novel mutations, Q254X and c.1363delC, were found. In total, 81.8% of the patients were from southern China. All the patients had xanthoma, and the average TC and LDL-C levels were 16.8 and 14.4 mmol/L, respectively. Echocardiography showed that 63.6% of the patients had aortic calcification, and 54.5% had mild regurgitation of the aortic valve. The coronary flow velocity reserve had a mean value of 2.12, and the cIMT was 0.17 cm. The follow-up period was between 3 months and 8 years. Although all the patients began the lipid-lowering treatment, 2 patients died because of severe cardiovascular disease. The LDL-C levels of 6 patients were slightly decreased by approximately 21% and remained far from the target values, and the other 3 patients' LDL-C levels increased by 13%. CONCLUSIONS: The results suggest that younger HoFH patients with W483X mutations had a severe phenotype and should receive more aggressive treatment.

Tripchlorolide Attenuates ß-amyloid Generation via Suppressing PPARγ-Regulated BACE1 Activity in N2a/APP695 Cells.

Due to its apparent rate-limiting function, BACE1 (ß-secretase) appears to be a prime target for prevention of amyloid-ß (Aß) generation in brains with Alzheimer's disease (AD). The activity of BACE1 is regulated by peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor binding site of the BACE1 promoter, indicating that PPARγ may be a potential target for AD treatment. Several studies have demonstrated that PPARγ activation is involved in the immunostimulation of amyloid-ß precursor protein processing by nonsteroidal anti-inflammatory drugs (NSAIDs). The present study found that tripchlorolide (T4), with a similar chemical structure to that of NSAIDs, decreased the levels of Aß secreted in N2a-APP695 cells. T4 treatment reduced the mRNA and protein levels of BACE1 and the protein level of sAPPß, a cleaved N-terminal fragment of APP by BACE1. The treatment also translocated PPARγ from cytoplasm to nuclear. Intriguingly, T4, like pioglitazone (a PPARγ agonist), suppressed the BACE1 activity in N2a-APP695 cells, which was attenuated by GW9662 (a PPARγ antagonist). These results indicate that T4 may be a PPARγ agonist to enhance the binding of nuclear PPARγ to the BACE1 promoter, which may in turn inhibit the transcription and translation of BACE1, suppress the activity of BACE1, and ultimately attenuate the generation of Aß. Due to its capability to alter Aß generation and to protect central neural system against the neurotoxicity of Aß, T4 may serve as a promising agent in modulating Aß-related pathology in Alzheimer's disease.

[Effects of exendin-4 on methylglyoxal-induced oxidative stress in PC12 cells].

OBJECTIVE: To study whether Exendin-4(Ex-4) could influence oxidative stress in PC12 cells induced by methylglyoxal and its underlying mechanism. METHODS: PC12 cells were cultured with methylglyoxal (0,0.25,0.50,0.75,1.0,2.0 mmol/L) for 12~48 h, or PC12 cells were pretreated with Ex-4 (25, 50, 100, 200 nmol/L) for 24 h then incubatedwith methylglyoxal (0.75 mmol/L) for 24 h. MTT assay was used to measure cell viability. Fluorescent probe method was used to detect reactive oxygen species (ROS) expression. Xanthine oxidase method was used to detect superoxide dismutase (SOD)activity. With pretreatment of Exendin-4 (100 nmol/L) for 24 h,the expressions ofP-IκBα, Inhibitor of NF-κB-α IκBα were detected by Western blot after PC12 cells were exposed to methylglyoxal (0.75 mmol/L) for 1 h. RESULTS: Following methylglyoxal administration, cell viability was gradually decreased in a dose-and time-dependent manner. Pretreatment with Ex-4 for 24 hours, cellviability were gradually increased compared with methylglyoxal-alone group. Pretreatment with Ex-4 (100 nmol/L) for 24 hours, ROS expression was reduced by65.30% (P<0.01) compared with methylglyoxal-alone group, ROS expression in NAC-pretreatment group was reduced by 107.40% (P<0.01); SOD activity in the Ex-4 pretreatment group was increased by 5.30 U/mg prot (P<0.01), SOD activity in the NAC pretreatment group was increased by 8.53 U/mg prot (P<0.01);the ratio of P-IκB-α/IκB-α in the Ex-4 pretreatment group was reduced by 25.50% (P<0.01), the ratio of P-IκB-α/IκB-α in the NAC pretreatment group was reduced by 35.14% (P<0.01). CONCLUSIONS: This study demonstrates that Ex-4 can increase the viabilities of PC12 cells and protect PC12 cells from oxidative stress induced by methylglyoxal, the mechanism may involve in suppressing the activation of protein IκB-α.

Receptors for advanced glycation end products (RAGE) is associated with microvessel density and is a prognostic biomarker for clear cell renal cell carcinoma.

The receptor for advanced glycation end products (RAGE) is involved in a variety of biological processes, including tumorigenisis. Previous studies have demonstrated that RAGE regulates the neo-angiogenesis related downstream molecule - vascular endothelial growth factor receptor 2 (VEGFR-2). Here, we investigated the potential relationship between RAGE, VEGFR-2 and angiogenesis in 80 renal cell carcinoma (RCC) patients. Real-time quantitative PCR and ELISA analysis were used to explore the RAGE and VEGFR-2 gene expression levels and the protein of VEGFR-2 expression. Meanwhile, angiogenesis was detected by the semi-quantification of endothelial cell marker CD34 combined with caldesmon, which was detected by microvessel density (MVD) technique and immunohistochemistry. Tumors were classified as low or high RAGE-expressing using the median as the cut-off. Immunofluorescence staining for RAGE protein was performed as well. Additionally, the median gene expression levels of VEGFR-2 in the tumors were significantly lower expressing low levels of RAGE expression, 0.34 (95% CI, 0.28-0.39) compared to the expressing high levels of RAGE expression, 0.45 (95% CI, 0.29-0.61), (P=0.03). The median MVD was significantly lower in the tumors expressing low levels of RAGE, 6.5 (95% CI, 6.21-7.43), compared to the expressing high levels, 7.9 (95% CI, 6.25-8.93), (P<0.01). Further, a positive association was certified with VEGFR-2 protein levels, P=0.07. Besides, RCC with high levels of RAGE expression are associated with high VEGFR-2 mRNA/protein levels and a higher density of microvessels; conversely, Kaplan-Meier survival analysis suggests that a significant correlation of elevated RAGE expression with decreased overall survival and metastasis-free survival. Our results establish that RAGE was identified as a potential prognostic biomarker for disease prognosis of RCC.

Identification of the gene defect responsible for severe hypercholesterolaemia using whole-exome sequencing.

Familial hypercholesterolaemia (FH) is a serious genetic metabolic disease. We identified a specific family in which the proband had typical homozygous phenotype of FH, but couldn't detect any mutations in usual pathogenic genes using traditional sequencing. This study is the first attempt to use whole exome sequencing (WES) to identify the pathogenic genes in Chinese FH. The routine examinations were performed on all parentage members, and WES on 5 members. We used bioinformatics methods to splice and filter out the pathogenic gene. Finally, Sanger sequencing and cDNA sequencing were used to verify the candidate genes. Half of parentage members had got hypercholesterolaemia. WES identified LDLR IVS8[-10] as a candidate mutation from 222,267 variations. The Sanger sequencing showed proband had a homozygous mutation inherited from his parents, and this loci were cosegregated with FH phenotype. The cDNA sequencing revealed that this mutations caused abnormal shearing. This mutation was first identified in Chinese patients, and this homozygous mutation is a new genetic type of FH. This is the first time that WES was used in Chinese FH patients. We detected a novel genetic type of LDLR homozygous mutation. WES is powerful tools to identify specific FH families with potentially pathogenic gene mutations.

[Establishment of a scoring system for predicting the positive rate of prostatic biopsy for prostate cancer].

OBJECTIVE: To identify the predictors of the positive results of transrectal ultrasound (TRUS)-guided biopsy for prostate cancer. METHODS: We performed univariate and multivariate logistic regression analyses on the relevant data on 385 male patients that underwent TRUS-guided biopsy for prostate cancer, including such potential predictors as age, body mass index (BMI), symptoms, results of digital rectal examination (DRE), tPSA, fPSA, free/total PSA ratio (f/tPSA), prostate volume (PV), and PSA density (PSAD) for identification of the risk factors related to the positive rate of biopsy. Then we constructed a scoring system as a tool for predicting prostate cancer in repeat biopsies and determined the sensitivity of the system by calculating the false positive rate using the receiver operating characteristic curve. RESULTS: Among the 385 patients, 139 (36.1%) were diagnosed with prostate cancer. On multivariate analysis, age (P < 0.01), DRE (P < 0.01), tPSA (P < 0.01), fPSA (P < 0.01), f/tPSA (P < 0.01), PV (P < 0.01), and PSAD (P < 0.01) were all significant predictors of prostate cancer. Multivariate logistic regression analysis showed age, tPSA, f/tPSA, PV, and PSAD to be independent predictors, with ORs and 95% CIs of 1.07 (1.05-1.16), 1.05 (1.02-1.15), 0.97 (0.86-0.99), 0.98 (0.87-0.96), and 1.79 (1.48-2.06), respectively. Moreover, patients with the risk score of 3-5 had a significantly higher rate of prostate cancer than those with 0-2 (64% vs 11%, P < 0.001). CONCLUSION: The scoring system on the key predictors of prostate cancer can help urologists to identify the men in need of prostatic biopsy.

Pre-clinical evaluation of AZD-2014, a novel mTORC1/2 dual inhibitor, against renal cell carcinoma.

Here we found that dual mTORC1/2 inhibitor AZD-2014 significantly inhibited RCC cell survival and growth, with higher efficiency than conventional mTORC1 inhibitors rapamycin and RAD001. RCC cell apoptosis was also induced by AZD-2014. AZD-2014 disrupted mTORC1/2 assembly and activation, while downregulating HIF-1α/2α and cyclin D1 expressions in RCC cells. Meanwhile, AZD-2014 activated autophagy, detected by p62 degradation, Beclin-1/ATG-5 upregulation and light LC3B-I/-II conversion. Autophagy inhibition by pharmacologic or siRNA-based means increased AZD-2014 activity in vitro, causing substantial RCC cell apoptosis. In vivo, AZD-2014 was more efficient than RAD001 in inhibiting 786-0 xenografts and downregulating HIF-1α/2α or p-AKT (Ser-473). Finally, AZD-2014's activity in vivo was further enhanced by co-administration of the autophagy inhibitor 3-methyaldenine. We provide evidence for clinical trials of using AZD-2014 in RCC treatment.